This invention relates to methods of preparing bone marrow stromal cells for use in ex vivo gene therapy.
Bone marrow is a complex and dynamic organ system comprised of hematopoietic cells, bone marrow stromal cells, and extracellular matrix. Pluripotent stem cells within the bone marrow proliferate and differentiate into numerous cell types including erythrocytes and leukocytes. It has been known for some time that association between stem cells and stromal cells is critical for this process. Studies in cell culture have shown that a layer of adherent stromal cells must be established before hematopoietic stem cells can grow and differentiate.
Bone marrow stromal cells are a heterogeneous population of cells that are defined by their morphology and function. In cell culture, they have a characteristic, spindle-shaped morphology and secrete growth factors and components that form an extracellular matrix. Stromal cells have been shown to divide in culture in response to epidermal growth factor (EGF; Kimura et al., 1988, Br. J. Hematol. 69:9–12), platelet derived growth factor (PDGF; Kimura et al., supra), and basic fibroblast growth factor (bFGF; Kimura et al., supra; Oliver et al., 1990, Growth Factors, 3:231–236).
Bone marrow and other biological substances can be frozen according to precalculated time and temperature curves (see, e.g., U.S. Pat. Nos. 4,107,937 and 4,117,881). Cryopreserved materials can be stored for extended periods of time with little degradation (Motta, M. R., (1993), Bone Marrow Trans. 12(2):177). Further, U.S. Pat. No. 4,963,489 indicates that when a suspension of fresh bone marrow and blood is mixed with cryoprotectants and frozen using computerized cryotechnological equipment, 90% of the cells remain viable when thawed. See also, Boswell et al., 1983, Exp. Hematol. 11:315–323; Gilabert et al., 1994, Eur. J. Haematol. 53:93–99; and Rowley, 1992, J. Hematother. Fall:1(3):233–250.
Bone marrow transplantation is a promising therapy for a number of diseases that involve hematopoietic cells. Transplantation can serve to replace cells that have been damaged by an intrinsic disease, such as an anemia, or in instances where hematopoietic cells have been destroyed by chemotherapy or radiation therapy. Transplantation can be autologous, i.e., the patient can serve as his or her own donor. Alternatively, a patient could receive bone marrow from a histocompatible donor. To date, however, conditions for preserving bone marrow, particularly bone marrow stromal cells, which could be used in numerous gene therapies, have not been optimized.
A major obstacle to gene therapies based on the modification of stromal cells is the procurement and sustained availability of therapeutically useful numbers of stromal cells. Consequently, despite the success of bone marrow transplantation, gene therapies that require successful transplantation of bone marrow stromal cells have not yet been realized.